The clinical utility of alpha2 adrenergic agonists for the anesthetic management of the surgical patient has been validated. Now the administration of alpha2 agonists is being considered for the management of chronic pain and for prolonged sedation of mechanically-ventilated patients. However, animal studies reveal that the analgesic and sedative/hypnotic effects of alpha2 adrenergic agonists diminish over time, a biologic phenomenon known as tolerance. The work proposed in this grant application seeks to define the mechanisms responsible for the induction and expression of tolerance to these two behavioral properties of alpha2 adrenergic agonists. Rats will be rendered tolerant to the hypnotic and/or analgesic properties of dexmedetomidine, a potent and highly-selective alpha2 agonist, and the function of key molecular components in a putative "tolerance cascade" will be determined in tissue obtained from the locus coeruleus (site of hypnotic action) and the spinal cord (site of analgesic action). The tolerance "phenotype" will also be studied in a cell culture paradigm using NG 108-15 cells to define the molecular mechanisms more directly. We propose that the putative tolerance cascade induced through the participation of the NMDA-type glutamate receptor complex and nitric oxide synthase (NOS) while the long-lasting expression of tolerance involve cAMP-dependent protein kinase (PKA) and it phosphorylated substrates, L-type Ca2+ channel (LTCC) and cAMP- responsive element binding protein (CREB). The expression of LTCC is induced by phosphoCREB and phosphorylated LTCC becomes facilitated an relatively resistant to blockade by alpha2 agonists, a key step in the transduction of the behavioral response to alpha2 agonists. We will determine whether the individual components are necessary for the development of tolerance by assessing its expression following chronic exposure to alpha2 agonists +/- blockade of the individual components. By repeating the biochemical analysis of the pivotal molecular components in the presence of the blocker we will establish the sequential relationship of the "blocked" component to the other molecular components within the tolerance cascade. To demonstrate whether a functionally-altered molecular component is sufficient to produce tolerance, we will mimic the biochemical alteration in naive paradigms and determine whether the tolerance phenotype can be reproduced. Data from these studies can be used to develop therapeutic strategies to prevent or reverse tolerance.